Knoevenagel Reaction over L-lysine/zeolite 4A Bull. Korean Chem. Soc. 2013, Vol. 34, No. 8 2367

http://dx.doi.org/10.5012/bkcs.2013.34.8.2367

Immobilization of L-Lysine on Zeolite 4A as an Organic-Inorganic Composite Basic

Catalyst for Synthesis of α,β-Unsaturated Carbonyl Compounds under

Mild Conditions

Farzad Zamani,†,‡,* Mehdi Rezapour,†,§ and Sahar Kianpour‡

†Department of Chemistry, Islamic Azad University, Shahreza Branch, Shahreza 31186145, Isfahan, Iran*E-mail: fzamani@iaush.ac.ir

‡Central Laboratory Complex, Sheikh-Bahai Department, Isfahan Science and Technology Town,

Isfahan University of Technology, Isfahan 8415683111, Iran§Department of Chemistry, Isfahan University of Technology, Isfahan 84156, Iran

Received February 23, 2013, Accepted May 16, 2013

Lysine (Lys) immobilized on zeolite 4A was prepared by a simple adsorption method. The physical and

chemical properties of Lys/zeolite 4A were investigated by X-ray diffraction (XRD), FT-IR, Brunauer-

Emmett-Teller (BET), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and

UV-vis. The obtained organic-inorganic composite was effectively employed as a heterogeneous basic catalyst

for synthesis of α,β-unsaturated carbonyl compounds. No by-product formation, high yields, short reaction

times, mild reaction conditions, operational simplicity with reusability of the catalyst are the salient features of

the present catalyst.

Key Words : Heterogeneous basic catalyst, Lys/zeolite 4A, Organic-inorganic composite, Knoevenagel reac-

tion, Solvent-free condition

Introduction

α-Amino acids are critical to life because they constitute

the building blocks of proteins and biopolymers carrying out

the most diverse functions in organisms. The physiological

importance of α-amino acids ensures a sustained interest in

their chemistry and properties, particularly in potential ap-

plications in many frontiers of modern materials science

including biocatalysis,1-3 drug delivery,4 and biodegradable

plastics industry.5 In addition, these compounds have receiv-

ed much attention because of their advantages from an

environmental as well as a resource standpoint.6-8

The immobilization of amino acids and other proteins into

inorganic host materials has attracted noticeable attention

over the last decade. In terms of catalytic purposes, it is

reasonable to assume that immobilization of amino acids on

a suitable support is a prerequisite for the use of catalysts in

large-scale processing. The development of novel bio-

catalyst would be in particular useful for the production of

pharmaceuticals and fine chemicals. The main challenge is

to design functional biocompatible materials and interfacial

structures, which allow stable attachment of amino acids

while maintaining their activity and function as close as

possible to their native state. The adsorption immobilization

is proposed to consist of the cationic form of the amino acid

attached to the negatively charged inorganic host surface.

Moreover, these electrostatic interactions are complemented

by hydrophobic interactions probably between neighboring

adsorbate molecules.9 In order to increase the efficiency of

amino acid immobilization and catalytic processes, it is

highly desirable to develop materials that have a large surface

area, hydrophilic character, high porosity, increased stability

with changes in the microenvironment, and high mechanical

strength. The adsorption behavior of amino acids on the

surfaces of materials such as hydroxyapatite, zirconium

phosphate modified silica, silica-gels, activated carbon and

zeolite has been investigated.10-16 Although many cases of

successful immobilization using inorganic porous materials

have been reported, most of the research published only uses

standard activity assays to monitor the catalytic performance.

In other words, the application of amino acids adsorbed on

inorganic supports in terms of catalytic purposes has receiv-

ed very little attention. Therefore, demonstration of their

utility including the possibility to reuse the catalyst is of

imminent importance.

Zeolites are the most favorable host materials due to

highly ordered pore systems, channels and cages of different

dimensions and shapes and the surface with negatively

charge-balanced with exchangeable cations.17,18 Zeolite NaA

(4A) is a synthetic zeolite with very small pores. It has a

three dimensional pore structure which is composed of soda-

lite cages, but connected through double four-membered

rings (D4R) of [SiO4]4− and [AlO4]

5−. The aluminosilicate

framework is anionic with large channels of spherical voids.

The principal channels have eight-oxygen (sixteen-member-

ed) rings as their minimum constriction. These eight-oxygen

rings are shared by the large cavities and allow material to

pass to the interior of a zeolite crystal.19 Due to its low cost

and high thermal stability, A-type zeolite has potential

applications in separation and catalysis processes.20

2368 Bull. Korean Chem. Soc. 2013, Vol. 34, No. 8 Farzad Zamani et al.

One of the most favorable interests in organic synthesis,

especially in the synthesis of fine chemical products such as

pharmaceuticals, is the facile synthesis of new carbon-

carbon bonds. Knoevenagel reaction is one of the different

types of C-C bond-forming reactions. It is a cross-aldol

condensation reaction between a carbonyl group and a meth-

ylene-activated substrate for synthesis of α,β-unsaturated

compounds. The catalysts traditionally used for this reaction

are ammonia, or primary or secondary amines.21 In recent

years, a wide range of catalysts and promoters such as

NbCl5,22 HClO4-SiO2,

23 CeCl3·7H2O/NaI,24 zeolities,25,26

TMSCl27 and ionic liquids 28-30 have been employed to

catalyze this reaction in solution or under solvent-free condi-

tions. In spite of the aforementioned efforts for the synthesis

of α,β-unsaturated compounds, many of these methods

involve expensive reagents, strong acidic/basic conditions,

long reaction times, low yields and use of toxic organic

solvents, reagents or catalysts. Therefore, to avoid these

limitations, the discovery of a new and efficient promoter or

catalyst with high catalytic activity, short reaction time,

recyclability and simple workup for the preparation of tri-

substituted alkenes under mild and practical conditions is of

prime interest.

In continuing our study to develop new efficient hetero-

geneous basic catalysts on inorganic porous materials,31-33

herein, we will present the heterogenization of lysine on

pure zeolite 4A material using a simple adsorption method,

which is a simple and eco-environmental friendly organic-

inorganic composite basic catalyst for the Knoevenagel

condensation of various aldehydes with ethyl cyanoacetate

under mild and heterogeneous conditions. To the best our

knowledge, noticeable work as to basic reactions such as

Knoevenagel condensation by á-amino acids adsorbed on

inorganic porous materials has not been reported yet.

Experimental

Materials. DL-Lysine monohydrochloride (> 98%, TLC),

tetraethyl orthosilicate (TEOS, 98%), tri-block copolymer

poly(ethylene glycol)-block-poly(propylene glycol)-block-

poly(ethylene glycol) (Pluronic P123, molecular weight =

5800, EO20PO70EO20), aluminum tri-sec-butylate (97%) and

2,4-pentandione (H-acac) were purchased from Aldrich.

Clinoptilolite was obtained from deposits of the Semnan

Region, Iran. The sample was ground and only particles

smaller than 71 μm (200 mesh) were used for the experi-

ment. Zeolite 4A was provided by Fujian Risheng Chemical.

All the reagents used were purchased from Merck in analy-

tical grade quality and further purification was not conduct-

ed.

Catalyst Preparation. Mesoporous silica SBA-15 through

the addition of H3PO4 was prepared as described in the

literature.34 2 g of Pluronic P123 were dissolved at room

temperature in 4.16 mL of H3PO4 (85%) and deionized

water (75.4 mL), then TEOS (4.6 mL) was added to this

solution and the synthesis was carried out by stirring at 35 oC

for 24 h in sealed teflon breakers and subsequently placed at

100 oC for 24 h. Then the solution was filtered, washed with

deionized water, and finally dried at 95 oC for 12 h in air.

Template removal was performed by calcination in air using

two successive steps, first heating at 250 oC for 3 h and then

at 550 oC for 4 h.

SiO2-Al2O3 was also used as the support, which was

prepared with the optimized Al/Si molar ratio, described in

the previous works.35 Aluminum tri-sec-butylate (97%) and

tetraethyl orthosilicate (98%) were used as the precursors,

and 2,4-pentandione (H-acac) as the complexing agent.

Appropriate amounts of Aluminum tri-sec-butylate and

tetraethyl orthosilicate were dissolved in the solvent, n-

butanol. The solution was heated to 60 oC. The components

were thoroughly mixed. Then the solution was cooled down

to room temperature, and H-acac as the complexing agent

was added. This clear solution was hydrolyzed with deioniz-

ed water (11.0 mol of H2O/mol of alkoxide). The solutions

were left overnight to hydrolyze the alkoxides, yielding

transparent gels. The transparent gels were dried at 110 oC to

remove water and solvent, and then calcined at 500 oC for 5

h to remove the organics.

Lysine adsorption on the different absorbents was carried

out according to the procedure previously reported.36 A

series of L-Lysine (Lys) amino acid solutions was prepared

by dissolving different amounts of amino acid in deionized

water. Afterwards, in each adsorption experiment, 50 mg of

the different adsorbents was suspended in 10 mL of the

amino acid solution. The pH of the solutions was adjusted

prior to mixing using 0.1 M NaOH or 0.1 M HCl. The result-

ing mixture was continuously shaken in a shaking bath with

a speed of 120 rpm at room temperature for 24 h. Finally, the

catalyst was separated from the solution by centrifugation.

The adsorption amount of amino acid was determined by

measuring the change of the amino acid concentration

before and after adsorption using UV-vis spectrophotometer

at λmax of lysine, 210 nm. The obtained catalysts were donat-

ed as Lys/zeolite 4A, Lys/Al2O3, Lys/SiO2-Al2O3 and Lys/

SBA-15.

Apparatus. The obtained materials were characterized by

X-ray diffraction (Bruker D8ADVANCE, Cu Kα radiation),

FT-IR spectroscopy (Nicolet 400D in KBr matrix in the

range of 4000-400 cm−1), Brunauer-Emmett-Teller (BET)

specific surface areas and Barrett-Joyner-Halenda (BJH) pore

size distribution (Series BEL SORP 18, at 77 K), scanning

electron microscopy (SEM) using Philips XL30 with SE

detector, UV-Vis spectroscopy (Varian, Cary 300) and Trans-

mission electron microscopy (TEM) using Phillips CM10

microscope.

General Procedure for Knoevenagel Condensation Reac-

tion. In a typical procedure, a mixture of benzaldehyde (2

mmol), ethyl cyanoacetate (2 mmol) and modified Lys/

zeolite 4A (0.1 g) was placed in a round bottom flask. The

suspension was agitated at room temperature for 10 minutes.

Completion of the reaction was monitored by TLC, using n-

hexane/ethyl acetate (5:1) as eluent. For the reaction work-

up, the catalyst was removed by filtration and washed with

hot ethanol. Then, the solvent was evaporated and a pure

Knoevenagel Reaction over L-lysine/zeolite 4A Bull. Korean Chem. Soc. 2013, Vol. 34, No. 8 2369

product was obtained. The products were identified using1H-NMR, 13C-NMR and FT-IR spectroscopy techniques.

Quantitative analyses were conducted with an Agilent 6820

GC equipped by gas chromatography (GC) using a HP 5890

Series II Chromatograph fitted with a packed column (18%

Carbowax 20 M/Chromosorb W AW-DMCS), equipped with

an HP 5971 Mass Selective Detector.

Results and Discussion

Catalyst Characterization.

XRD: The XRD patterns of zeolite 4A and Lys/zeolite 4A

are presented in Figure 1. Zeolite 4A depicts several sharp

characteristic peaks at 2θ values of 7º, 10.4º, 12.8º, 16.5º,

21.6º, 24º, 26.2º, 27º, 30º, 31º, 31.1º, 32.5º, 33.5º and 34.3º

that has been reported in the literature,37 which can be

indexed to the (200), (220), (222), (420), (440), (600), (622),

(640), (642), (694), (600), (840), (842) and (664) reflections

of the cubic crystalline system and the space group Fm3c.38

The zeolite 4A and Lys/zeolite 4A samples show the same

pattern, indicating that the structure of the zeolite 4A is well

retained even after immobilization with lysine. However, the

intensity of the characteristic reflection peaks of the Lys/

zeolite 4A (2θ = 7-45) sample is found to be reduced (Fig.

1), which is possibly due to the presence of guest moieties

onto the framework of zeolite 4A, resulting in the decrease

of crystallinity.

FT-IR: The FT-IR spectra of zeolite 4A, L-lysine and

optimized lysine-supported sample are shown in Figure 2. In

the FT-IR spectrum of the zeolite 4A, the characteristic

bands for zeolite framework at 565 cm−1 due to the external

vibration of double four-rings, 1010 cm−1 for the internal

vibration of (Si, Al)-O asymmetric stretching, 688 cm−1 for

the internal vibration of (Si, Al)-O symmetric stretching, and

470 cm−1 for the internal vibration of (Si, Al)-O bending

were observed (Fig. 2(a)). The board band at around 3100-

3600 cm−1 and the sharp one at 1665 cm−1, which are related

to OH, also appeared.39-42 In terms of Lys/zeolite 4A, the

presence of peaks at about 2900 cm−1 is attributed to the

aliphatic C-H stretching of the amino acid, which is at low

intensity because of the very broad band of zeolite (Fig.

2(c)). These are in accordance with the spectrum of Lys (Fig.

2(b)). The bands at 1418 cm−1, 1595 cm−1 and 1519 cm−1,

and are respectively assigned to COO−as, COO−

as plus NH+3

sd

(α-amino group) and NH+3sd (side-chain amino group).43

Furthermore, the FT-IR spectrum of Lys/zeolite 4A has no

an absorption band at 1733 cm−1 (C=Oas) which indicates

that the α-carboxyl group is completely present in the de-

protonated form (COO) (Fig. 2(c)). Hence, the FT-IR spectro-

scopic analysis exhibits that the major fraction of lysine

adsorbed onto the silica surface is in the cationic state.43

Kitadai has reported that if lysine adsorbs on montmori-

llonite through the α-amino group, both of the characteristic

bands of α-amino group and side-chain amino group are

likely shift to lower wavenumbers,44 which similarly did not

occur in Lys/zeolite 4A. Therefore, it might be deduced that

lysine adsorbed on zeolite 4A through the protonated side-

chain amino group but not through the protonated α-amino

group.

N2 Adsorption-Desorption Isotherms. The specific surface

area and the pore size of the samples are summarized in

Table 1. It can be seen that the pore volume and the surface

area of zeolite 4A decreased drastically after Lys adsorptionFigure 1. The powder XRD pattern of (a) zeolite 4A and (b) Lys/zeolite 4A.

Figure 2. FT-IR spectra of (a) zeolite 4A, (b) lysine and (c) Lys/zeolite 4A.

2370 Bull. Korean Chem. Soc. 2013, Vol. 34, No. 8 Farzad Zamani et al.

from 210.4 to 113.7 m2 g−1 and 0.22 to 0.17 cm3 g−1 respec-

tively. This large reduction in the specific pore volume and

the specific surface area could be attributed to the tight

packing of Lys molecule inside the pore channels of zeolite

4A. On the other hand, the data for the Lys/SiO2-Al2O3 and

Lys/zeolite 4A samples indicated a growth in the average

pore diameter in comparison to Al2O3-SiO2 and zeolite 4A.

The results are likely attributed to the presence of lysine in

the structure of catalyst.

SEM: Figure 3 shows the SEM images of the zeolite 4A

and Lys/zeolite 4A samples. These figures reveal that Lys/

zeolite 4A (Fig. 3(c), (d)) has approximately the same

morphology as commonly observed in zeolite 4A (Fig. 3(a),

(b)), which can be described as truncated-side cubic crystals

with a size in the range 1-2 µm. This finding indicates that

there is no change in the morphology of silica after being

modified with the amino acid. As a result, N2 adsorption and

SEM image of the samples after lysine adsorption reveal that

the amino acid molecule is packed inside the pore channels

of zeolite 4A adsorbent.

TEM: The TEM micrograph of Lys/zeolite 4A is depicted

in Figure 4. It can be seen that the sample has uniform

particle size of about 100-200 nm, and the places with darker

contrast could be assigned to the presence of lysine with

different dispersion, probably located into the support pores,

which is in accordance with the SEM images, BET and

XRD results.

Effect of Operational Parameters on the Lysine Ad-

sorption. The adsorption of lysine has been studied under a

range of solution conditions, e.g., pH, amino acid concen-

tration, contact time and different supports, to reach an

optimum condition.

In order to find out the adsorption capacity of adsorbents,

the Lys adsorption isotherms of five different adsorbent

materials, i.e. clinoptilolite, Al2O3, SiO2-Al2O3, zeolite 4A

and SBA-15, have been determined at different buffer solu-

tion pH ranging from 5 to 10 (Fig. 5). In general, there are

four different dissociation states of lysine in an aqueous

media, including di-cationic, cationic, zwitterionic, and anionic,

which are mainly dependent on solution pH.45 It means that

as pH increases, the dissociation state changes from di-

cationic to anionic form. Near the isoelectric point of lysine

(9.7), although the zwitterionic state is the major dissocia-

tion state of dissolved lysine, it has been revealed that the

Table 1. Physical properties of the samples

Sample

BET surface

area

(m2 g−1)

Pore volume

(cm3 g−1)

Average pore

diameter

(nm)

SBA-15 1400 1.90 9

Clinoptilolite 20.8 0.03 0.61

Zeolite 4A 210.4 0.22 0.73

Al2O3 330 0.25 5

SiO2-Al2O3 498 0.46 4.5

Lys/Zeolite 4A 113.7 0.17 0.95

Figure 3. Scanning electron microscopy (SEM) photographs of(a,b) zeolite 4A and (c,d) Lys/zeolite 4A.

Figure 4. Transmission electron microscopy (TEM) image of Lys/zeolite 4A.

Figure 5. Lysine adsorption isotherms on various porousadsorbents at different pH conditions (concentration of lysine = 10mmol L−1, 24 h, room temperature).

Knoevenagel Reaction over L-lysine/zeolite 4A Bull. Korean Chem. Soc. 2013, Vol. 34, No. 8 2371

major fraction of lysine adsorbed onto the adsorbent surface

is in the cationic state.43,44 It can be seen that there is an

upward trend in the amount of Lys adsorbed onto all the

adsorbents over the pH range of 5 to nearly 10, reaching a

point of maximum lysine adsorption at pH 10, which is close

to the isoelectric point of lysine (9.7). It has been shown in

some studies that proteins tend to adsorb onto surfaces more

via strong electrostatic interactions between the surface and

the protein.46 In the pH range of 5 to nearly 10, the amount

of adsorbed cationic lysine swiftly increases because the

surface becomes more negatively charged, whereas the mole

fraction of cationic lysine in the solution do not substantially

change. As a result, the adsorption could be mainly driven

by the strong electrostatic interaction between the negatively

charged zeolite 4A surface and the positively charged cati-

onic lysine. Although the mole fraction of cationic lysine in

the solution decreases at high pH, the amount of adsorbed

cationic lysine still increases slowly. It can be explained that

the surface charge density of the negatively charged surface

increases faster than the mole fraction of cationic lysine

decreased in solution.47

It is also obvious from Figure 5 that the adsorption

capacities of the different adsorbents have the following

order: zeolite 4A > SiO2-Al2O3 > clinoptilolite > Al2O3 >

SBA-15, although SBA-15 has higher surface areas as

compared to the other adsorbents (Table 1). In the case of

zeolite 4A, the presence of well-ordered pore system sup-

ports the diffusion of the Lys molecule into the interior part

of the pores. Moreover, the surface area of zeolite 4A is

higher than the clinoptilolite and provides the way for more

accessible adsorption sites. Therefore, the adsorption capa-

city of zeolite 4A is higher than clinoptilolite. By contrast,

although SBA-15 has a higher mesopore volume and ultra

large pore diameter, the Lys adsorption capacity is quite low.

This indicates that in terms of SBA-15, the surface charac-

teristic of the adsorbent plays an important role in deter-

mining the adsorption capacity. It has been previously

reported that the surface hydroxyl groups of SBA-15 is

around 4.1 OH groups per nm2.48 These surface hydroxyl

groups may prefer to form hydrogen bond with the water

molecules present in the buffer rather than the Lys molecules

and form a hydration sphere on the surface. This would

suppress the interaction between the Lys molecule and the

adsorbent surface. Therefore, the reduction in the adsorption

capacity is expected. Moreover, it is well known fact that the

pore curvature decreases with increasing pore diameter. The

reduced pore curvature also affects the interaction between

the neighbouring lysine molecules which reduces the tight

packing of the lysine molecules.14 As can be seen, SBA-15

has the highest pore diameter in comparison to the other

supports. As a result, although SBA-15 has the high acidic

site and pore volume, the low Lys adsorption capacity is

expected due to these aspects. In addition, According to the

data, it can be said that pore size do not affect the adsorption

of lysine on zeolites, which is in agreement with the previ-

ous works.36,49 Moreover, as lysine amino acid is hydrophilic,

the hydrophilicity/hydrophobicity of zeolitic materials, which

is affected by their Si/Al ratio, plays a key role in the ad-

sorption of amino acids onto the zeolite surface.49,50 It is as-

sumed that zeolites with high Si/Al ratio show hydrophobic

character.49 Compared with SBA-15, zeolite 4A and SiO2-

Al2O3 are rich in alumina, which leads to having hydrophilic

surface. As a result, the adsorption of lysine on these zeolites

is higher than SBA-15 due to the hydrophilicity. In addition,

in terms of zeolite 4A, the electrical imbalance arising from

the substitution of Si4+ by Al3+ in the structure of zeolites is

compensated by balancing cations held electrostatically

within the zeolite. These cations are not an integral part of

the zeolite Si/Al-O framework and are usually called ex-

changeable cations, since they are fairly mobile and readily

replaced by other cations. Therefore, these exchangeable

cations might improve the amino acid adsorption capacity of

zeolite 4A.

As a result, from the industrial point of view, since zeolite

4A is easily available among the solid-acid catalysts, it was

employed for the further steps.

In order to test the influence of amino acid concentration,

Lys adsorption was determined at six different amino acid

concentrations, ranging from 5 to 30 mmol L−1 at the pH 10

(Fig. 6). The amount of amino acid adsorption onto zeolite

4A significantly increased with raising initial concentration

of amino acid solution. This phenomenon can be explained

that when the amino acid concentration in the bulk is low,

the amino acid is randomly deposited on the adsorbent

surface. On the contrary, when the amino acid concentration

is high, functional group of the two or more amino acid side

chains approaches each other until they touch within their

van der Waals radii, and helps close packing of the amino

acid molecules.51,52 According to the results, 25 mmol L−1

was selected as the optimized Lys adsorption, which ex-

hibited the total adsorption of 2.65 mmol g−1 on the zeolite

4A surface.

The time dependence of lysine adsorption onto zeolite 4A

was investigated to determine the time required for equi-

librium to be reached between the solid and solution (Fig. 7).

Figure 6. Adsorption isotherm for lysine onto zeolite 4A (pH = 10,24 h, room temperature).

2372 Bull. Korean Chem. Soc. 2013, Vol. 34, No. 8 Farzad Zamani et al.

It is clear from the figure that the adsorbed amount increases

with contact time to reach a plateau of 2.65 mmol g−1 at 24 h.

Looking more closely at the trend for the lysine adsorption,

it can be seen that a slow reduction in adsorbed amount

begins from 72 h onwards, which could be attributed to a

loss of stability in the zeolite 4A. In other words, the porous

structure of zeolite 4A is likely degraded upon extended

exposure to water.

Adsorption Behavior of Lysine on Zeolite 4A. In general,

there are three types of surface hydroxyl groups in the

structure of zeolite 4A including Al-OH, Si-OH and Si-OH-

Al. The Brönsted acidity of the three hydroxyl protons

increases in the order Si-OH-Al > Al-OH > Si-OH, with the

bridging hydroxyl proton being the strongest acid.53-56 There-

fore, the basic media interacts with the bridging hydroxyl

protons more than the other protons and the surface of

zeolite 4A becomes more negatively charged. Hence, the

adsorption behavior can be explained by the electrostatic

interaction between the negatively charged surface and the

positively charged lysine. Another possibility increases in

the contribution of hydrophobic interaction between the

side-chain alkyl groups of neighboring adsorbing lysine with

increasing pH and amino acid concentration.57,58 As the

amount of adsorbed lysine increases, each adsorbed lysine

molecule approaches more closely together, and the ad-

sorbate-adsorbate interaction becomes stronger. As a result,

the hydrophobic interaction would complement the electro-

static interaction between lysine and zeolite 4A surface.

Hence, it might be expected that lysine has a strong adsorp-

tion on the zeolite 4A surface which would be suitable for

catalytic purposes.

Effect of Reaction Parameters on the Catalytic Activity.

The Knoevenagel condensation of benzaldehyde with ethyl

cyanoacetate was chosen as a model reaction to test the

catalytic activity of the Lys/zeolite 4A. Therefore, the effect

of various reaction parameters on the condensation of benz-

aldehyde with ethyl cyanoacetate has been studied using

Lys/zeolite 4A as a basic catalyst.

Different solvents such as water, acetonitrile, methanol

and dichloromethane were used for the Knoevenagel con-

densation over Lys/zeolite 4A. The solvent-free condition

was studied as well. The results are summarized in Table 2.

As mentioned earlier, according to the FT-IR spectroscopic

analysis, the major fraction of lysine adsorbed onto the silica

surface is in the cationic state. Hence, it could be expected

that the surface of the catalyst consists of both basic and acid

sites (NH3+ and COO). It can be seen that the highest yield

of 83% was obtained in the absence of solvent. Although the

reactants, intermediates and products are more stabilized in

polar solvents such as water,59 the catalyst showed a lower

activity in water compared with the solvent-free condition.

A reasonable explanation is that the amino acid adsorbed on

zeolite 4A can be flushed with water, which leads to pore

blocking as well as reducing the active sites of the catalyst. It

is also expected that the acid and base groups in Lys/zeolite

4A would be in equilibrium between the free acid-base and

the ion pair, which results from neutralization. Therefore, the

solvent could have a dramatic effect on this equilibrium. In

general, protic solvents would confer different properties to

aprotic solvents as a result of differing abilities to stabilize

the neutralized ion pair. The equilibrium would lie towards

the ion pair in protic solvents, e.g. methanol and water, as

proton exchange would be rapid and the protic solvent

would stabilize the ion pair the most. Aprotic solvents such

as dichloromethane and acetonitrile would cause slower

exchange of the protons and would stabilize the ion pair

much less than a protic solvent, forcing the equilibrium in

favor of the free acid and base.60

In order to investigate the importance of amount of the

catalyst, the Knoevenagel reaction between benzaldehyde

and ethyl cyanoacetate over Lys/zeolite 4A was investigated

Figure 7. Contact time (lysine concentration = 25 mmol g−1, pH =10, room temperature).

Table 2. Effect of solvent on the Knoevenagel condensation overLys/zeolite 4Aa

Solvent Yield (%)b

Methanol 58

Dichloromethane 75

Water 60

Acetonitrile 67

Solvent-free 83

aReaction conditions: catalyst (0.06 g), benzaldehyde (2 mmol), ethylcyanoacetate (2 mmol), room temperature, reaction time = 10 min, selec-tivity = 100%. bIsolated yield

Table 3. Effect of amount of catalyst on the Knoevenagel conden-sation over Lys/zeolite 4Aa

Catalyst amount (g) Yield (%)b

0.02 55

0.04 75

0.06 83

0.1 98

0.12 98

aReaction conditions: benzaldehyde (2 mmol), ethyl cyanoacetate (2mmol), solvent-free, room temperature, reaction time = 10 min, selec-tivity = 100%. bIsolated yield

Knoevenagel Reaction over L-lysine/zeolite 4A Bull. Korean Chem. Soc. 2013, Vol. 34, No. 8 2373

by different amounts of the catalyst (Table 3). It is observed

that while the amount of Lys/zeolite 4A increases from 0.02

to 0.1 g, the product yield rises significantly from 55% to

98%. It is because of the availability of more acid-base sites,

which favors the dispersion of more active species. After-

wards, the percentage of yield remains stable at 98% bet-

ween 0.1 g and 0.12 g.

In order to investigate effect of the support (zeolite 4A) on

the Knoevenagel reaction, lysine (with the same ratio of the

optimized catalyst) without any support as catalyst was used

in the Knoevenagel reaction of benzaldehyde by keeping

other parameters constant. After 24 h, approximately 75%

yield was observed. Therefore, the adsorption of lysine onto

the zeolite 4A (as a support) is a valuable way to improve the

catalytic activity of the amino acid for the Knoevenagel

reaction.

In the view of green chemistry, the reusability of the

catalyst was also studied by using Lys/zeolite 4A in recycl-

ing experiments (Table 4). In order to regenerate the catalyst,

after completion of the reaction, it was separated by

centrifuge and washed several times with deionized water.

Then, it was dried at 60 ºC and reused in the subsequent run.

By using regenerated sample after four cycles, the yield

decreased by 10% and the selectivity was constant (100%).

Application of Lys/zeolite 4A in Knoevenagel Conden-

sation of Various Aldehydes with Ethyl Cyanoacetate.

Our major concern was to examine the activity and selec-

tivity of prepared catalyst for basic reactions such as Knoev-

enagel condensation. Accordingly, a number of Knoevenagel

condensation reactions were carried out with a variety of

aromatic and aliphatic aldehydes with ethyl cyanoacetate

over Lys/zeolite 4A, after ascertaining the optimum experi-

mental conditions. The results are represented in Table 5. It

is notable that the aromatic aldehydes, having different

substituents such as chloro, nitro, methoxy and methyl, were

converted to the corresponding arylidene derivatives with

good to high yields. The aromatic aldehydes with electron-

withdrawing groups such as chloro and nitro proceeded at

faster rates than those with electron-donating groups such as

methoxy and methyl. These results showed that electron-

donating substituents in aromatic ring appear to retard the

rate of reaction due to inactivation of aldehyde group. In

addition, the Knoevenagel condensation was carried out

using aliphatic aldehydes, which represented the high pro-

duct yields. Interestingly, the results clearly established that

the obtained products were E-isomer forms.

Conclusion

In conclusion, our studies showed that the immobilization

of lysine amino acid onto zeolite 4A serve as highly active

organic-inorganic composite basic catalyst for the Knoev-

enagel reaction of various aromatic and aliphatic aldehydes

with ethyl cyanoacetate. The merit of this methodology is

that it is simple, fast, mild, and efficient. Therefore, we

believe that the reported catalyst could greatly contribute to

the environmentally greener and safer process.

Acknowledgments. Dr. C. Hyland (University of Wollon-

gong) is gratefully acknowledged for his helpful comments.

The authors also thank the Isfahan Science and Technology

Town for the support of this work. And the publication cost

of this paper was supported by the Korean Chemical Society.

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Fresh 98

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2 93

3 90

4 88

aReaction conditions: benzaldehyde (2 mmol), ethyl cyanoacetate (2mmol), solvent-free, room temperature, reaction time = 10 min, selec-tivity = 100%. bIsolated yield

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